In the special issue “Signaling Molecules and Signal Transduction in Cells” authors were invited to submit papers regarding important and novel aspects of extra- and intracellular signaling which have implications on physiological and pathophysiological processes. These aspects included compounds which are involved in these processes, elucidation of signaling pathways, as well as novel techniques for the analysis of signaling pathways. In response, various novel and important topics are elucidated in this special issue. Full article

Patients with solid cancer frequently develop bone metastases (BM). Zoledronic acid (Zometa^®, ZA), routinely used to treat patients with BM, acts on osteoclasts and also has antitumor properties. We aimed to assess the effect of ZA over time in novel bone turnover markers (RANK/receptor activator of nuclear factor-k B ligand (RANK-L)/ Osteoprotegerin (OPG)) and to correlate these with serum N-terminal telopeptide (NTX). The study prospectively evaluated levels of RANK, RANK-L and OPG transcripts by real-time PCR and NTX expression by ELISA in the peripheral blood of 49 consecutive patients with advanced breast, lung or prostate cancer. All patients received the standard ZA schedule and were monitored for 12 months. Median baseline values of RANK, RANK-L and OPG were 78.28 (range 7.34–620.64), 319.06 (21.42–1884.41) and 1.52 (0.10–58.02), respectively. At 12 months, the median RANK-L value had decreased by 22% with respect to the baseline, whereas median OPG levels had increased by about 96%. Consequently, the RANK-L/OPG ratio decreased by 56% from the baseline. Median serum NTX levels decreased over the 12-month period, reaching statistical significance (p < 0.0001). Our results would seem to indicate that ZA modulates RANK, RANK-L and OPG expression, thus decreasing osteoclast activity. Full article

The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points. Full article

Bone marrow derived human mesenchymal stem cells (hMSCs) show promising potential in regeneration of defective tissue. Recently, gene silencing strategies using microRNAs (miR) emerged with the aim to expand the therapeutic potential of hMSCs. However, researchers are still searching for effective miR delivery methods for clinical applications. Therefore, we aimed to develop a technique to efficiently deliver miR into hMSCs with the help of a magnetic non-viral vector based on cationic polymer polyethylenimine (PEI) bound to iron oxide magnetic nanoparticles (MNP). We tested different magnetic complex compositions and determined uptake efficiency and cytotoxicity by flow cytometry. Additionally, we monitored the release, processing and functionality of delivered miR-335 with confocal laser scanning microscopy, real-time PCR and live cell imaging, respectively. On this basis, we established parameters for construction of magnetic non-viral vectors with optimized uptake efficiency (~75%) and moderate cytotoxicity in hMSCs. Furthermore, we observed a better transfection performance of magnetic complexes compared to PEI complexes 72 h after transfection. We conclude that MNP-mediated transfection provides a long term effect beneficial for successful genetic modification of stem cells. Hence, our findings may become of great importance for future in vivo applications. Full article

The deubiquitinating enzyme USP14 has been identified and biochemically studied, but its role in lung cancer remains to be elucidated. The aim of this study was to evaluate the prognostic significance of USP14 in patients with lung adenocarcinoma and to define its role in lung cancer cell proliferation. USP14 mRNA levels in different non-small cell lung cancer (NSCLC) cell lines were detected by real-time qPCR. USP14 protein levels in surgically resected samples from NSCLC patients, and in NSCLC cell lines, were detected by immunohistochemistry or Western blot. The correlation of USP14 expression with clinical characteristics and prognosis was determined by survival analysis. After silencing USP14, cell proliferation was assessed by MTT assay and the cell cycle was measured by FACS assay. It was found that USP14 expression was upregulated in NSCLC cells, especially in adenocarcinoma cells. Over-expression of USP14 was associated with shorter overall survival of patients. Downregulation of USP14 expression arrested the cell cycle, which may be related to β-catenin degradation. Over-expression of USP14 was associated with poor prognosis in NSCLC patients and promoted tumor cell proliferation, which suggests that USP14 is a tumor-promoting factor and a promising therapeutic target for NSCLC. Full article

The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. We report that T-2 toxin significantly induces CYP1A4 and CYP1A5 expression in chicken embryonic hepatocyte cells. The enzyme activity assays of CYP1A4 and CYP1A5 heterologously expressed in HeLa cells indicate that only CYP1A5 metabolizes T-2 to 3'OH-T-2 by the 3'-hydroxylation of isovaleryl groups. In vitro enzyme assays of recombinant CYP1A5 expressed in DH5α further confirm that CYP1A5 can convert T-2 into TC-1 (3'OH-T-2). Therefore, CYP1A5 is critical for the metabolism of trichothecene mycotoxin in chickens. Full article

As olfactory receptor axons grow from the peripheral to the central nervous system (CNS) aided by olfactory ensheathing cells (OECs), the transplantation of OECs has been suggested as a plausible therapy for spinal cord lesions. The problem with this hypothesis is that OECs do not represent a single homogeneous entity, but, instead, a functionally heterogeneous population that exhibits a variety of responses, including adhesion and repulsion during cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. In this paper, we report a system based on modified OECs carrying magnetic nanoparticles as a proof of concept experiment enabling specific studies aimed at exploring the potential of OECs in the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) in vitro, their migration can be modulated by magnetic fields; and (iii) their transplantation in organotypic slices of spinal cord and peripheral nerve showed positive integration in the model. Altogether, these findings indicate the therapeutic potential of magnetized OECs for CNS injuries. Full article

The transition from α-helical to β-hairpin conformations of α-syn12 peptide is characterized here using long timescale, unbiased molecular dynamics (MD) simulations in explicit solvent models at physiological and acidic pH values. Four independent normal MD trajectories, each 2500 ns, are performed at 300 K using the GROMOS 43A1 force field and SPC water model. The most clustered structures at both pH values are β-hairpin but with different turns and hydrogen bonds. Turn_9-6 and four hydrogen bonds (HB_9-6, HB_6-9, HB_11-4 and HB_4-11) are formed at physiological pH; turn_8-5 and five hydrogen bonds (HB_8-5, HB_5-8, HB_10-3, HB_3-10 and HB_12-1) are formed at acidic pH. A common folding mechanism is observed: the formation of the turn is always before the formation of the hydrogen bonds, which means the turn is always found to be the major determinant in initiating the transition process. Furthermore, two transition paths are observed at physiological pH. One of the transition paths tends to form the most-clustered turn and improper hydrogen bonds at the beginning, and then form the most-clustered hydrogen bonds. Another transition path tends to form the most-clustered turn, and turn_5-2 firstly, followed by the formation of part hydrogen bonds, then turn_5-2 is extended and more hydrogen bonds are formed. The transition path at acidic pH is as the same as the first path described at physiological pH. Full article

The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect. Full article

In this study we found that licochalcone E (LicE), a recently isolated retrochalcone from Glycyrrhiza inflata, exhibits potent anti-inflammatory effects in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema and lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage models. Topical application of LicE (0.5–2 mg) effectively inhibited TPA-induced (1) ear edema formation; (2) phosphorylation of stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK), c-Jun, and extracellular signal regulated kinase 1/2; and (3) expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins in mouse skin. The treatment of RAW 264.7 cells with LicE (2.5–7.5 μmol/L) induced a profound reduction in LPS-induced (1) release of NO and prostaglandin E₂; (2) mRNA expression and secretion of interleukin (IL)-6, IL-1β and tumor necrosis factor-α; (3) promoter activity of iNOS and COX-2 and expression of their corresponding mRNAs and proteins; (4) activation of AKT, p38 mitogen activated protein kinase (MAPK), SAPK/JNK and c-Jun; (5) phosphorylation of inhibitor of κB (IκB) kinase-αβ and IκBα, degradation of IκBα, translocation of p65 (RelA) to the nucleus and transcriptional activity of nuclear factor (NF)-κB; and (6) transcriptional activity of activator protein (AP)-1. These results indicate that the LicE inhibition of NF-κB and AP-1 transcriptional activity through the inhibition of AKT and MAPK activation contributes to decreases in the expression of pro-inflammatory cytokines and the inducible enzymes iNOS and COX-2. Full article

In renal cell carcinoma (RCC), single members of the Wnt/β-catenin signaling cascade were recently identified to contribute to cancer progression. However, the role of Wnt1, one of the key ligands in β-catenin regulation, is currently unknown in RCC. Therefore, alterations of the Wnt1/β-catenin axis in clear cell RCC (ccRCC) were examined with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS). Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and β-catenin expression by immunohistochemistry in tissue microarrays. Expression scores, including intensity and percentage of stained cells, were compared between normal kidney and ccRCCs. Data was categorized according to mean expression scores and correlated to tumor and patients’ characteristics. Survival was analyzed according to the Kaplan-Meier and log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to explore the independent prognostic value of Wnt1 and β-catenin. In ccRCCs, high Wnt1 was associated with increased tumor diameter, stage and vascular invasion (p ≤ 0.02). High membranous β-catenin was associated with advanced stage, vascular invasion and tumor necrosis (p ≤ 0.01). Higher diameter, stage, node involvement, grade, vascular invasion and sarcomatoid differentiation (p ≤ 0.01) were found in patients with high cytoplasmic β-catenin. Patients with a high cytoplasmic β-catenin had a significantly reduced OS (hazard ratio (HR) 1.75) and CSS (HR 2.26), which was not independently associated with OS and CSS after adjustment in the multivariable model. Increased ccRCC aggressiveness was reflected by an altered Wnt1/β-catenin signaling. Cytoplasmic β-catenin was identified as the most promising candidate associated with unfavorable clinicopathology and impaired survival. Nevertheless, the shift of membranous β-catenin to the cytoplasm with a subsequently increased nuclear expression, as shown for other malignancies, could not be demonstrated to be present in ccRCC. Full article

Oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a major role in the antioxidant system and they also catalyze superoxide radicals (O₂^·−). Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of mouse tissue, SOD1 is essential for the maintenance of tissue homeostasis. To clarify the cellular function of SOD1, we investigated the cellular phenotypes of Sod1-deficient fibroblasts. We demonstrated that Sod1 deficiency impaired proliferation and induced apoptosis associated with O₂^·− accumulation in the cytoplasm and mitochondria in fibroblasts. Sod1 loss also decreased the mitochondrial membrane potential and led to DNA damage-mediated p53 activation. Antioxidant treatments effectively improved the cellular phenotypes through suppression of both intracellular O₂^·− accumulation and p53 activation in Sod1-deficient fibroblasts. In vivo experiments revealed that transdermal treatment with a vitamin C derivative significantly reversed the skin thinning commonly associated with the upregulated p53 action in the skin. Our findings revealed that intrinsic O₂^·− accumulation promoted p53-mediated growth arrest and apoptosis as well as mitochondrial disfunction in the fibroblasts. Full article

In this study, we developed a one step process to synthesize nanogel containing silver nanoparticles involving electron beam irradiation. Water-soluble silver nitrate powder is dissolved in the distilled water and then poly(acrylic acid) (PAAc) and hexane are put into this silver nitrate solution. These samples are irradiated by an electron beam to make the PAAc nanogels containing silver nanoparticles (Ag/PAAc nanogels). The nanoparticles were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). In addition, the particle size and zeta-potential were confirmed by a particle size analyzer (PSA). The antibacterial properties of the nanogels were evaluated by paper diffusion test. The Ag/PAAc nanogels had an antibacterial effect against Escherichia coli and Staphylococcus aureus. The nanogels also demonstrated a good healing effect against diabetic ulcer. The size of the Ag/PAAc nanogels decreased with increasing irradiation doses, and the absolute value of the zeta potential increased with increasing irradiation doses. Also, the Ag/PAAc nanogels exhibited good antibacterial activity against both Gram-negative and Gram-positive bacteria. In in vivo wound healing, the Ag/PAAc nanogels have a good healing effect. Full article

Objective: The aim of this study was to analyze the correlation and clinical significance between the expression of Mucin-1 (MUC1) and the Lewis y antigen with chemoresistance in ovarian epithelial cancers. Methods: Ovarian cancer patients (n = 92) treated at our hospital from May 2005 to July 2009 were divided, according to their treatment and follow-up outcomes, into a resistant group (n = 37) or sensitive group (n = 55). The expression of MUC1 and Lewis y antigen in ovarian cancer tissues was detected using immunohistochemistry and correlated with chemoresistance. Results: The positive rates of MUC1 and Lewis y antigen in the resistant group were both 91.89%, significantly higher than their positive rates in the sensitive group (65.45% and 69.09%, respectively, and both p < 0.05). MUC1 or Lewis y expression and the pathological stage of the tissue were independent risk factors for chemoresistance (all p < 0.05). Conclusion: The increased expression of MUC1 and the Lewis y antigen is a significant risk factor for chemoresistance in patients with ovarian epithelial cancer. Full article

Epithelial ovarian cancer (EOC) is one of the leading causes of cancer deaths in women worldwide. Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for sumoylation, regulates protein function and plays an important role in sumoylation-mediated cellular pathways. Although sumoylation plays a key role in DNA repair and tumorgenesis, whether Ubc9 is involved in EOC progression remains unknown. In the present study, we constructed Ubc-9 expressed recombined plasmid pEGFP-N1-Ubc9. The mRNA levels of Ubc9 were confirmed in human ovarian cell lines before and after transfection with pEGFP-N1-Ubc9 or small interfering RNA (siRNA) targeted Ubc9 by real-time polymerase chain reaction (PCR). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to observe the effect of Ubc9 on cell proliferation. The protein levels of Ubc9, and proliferation-related signals Akt and physphorylated Akt were determined by Western blot. Our results showed that proliferation of EOC cells increased significantly in Ubc9 overexpressing cells, but decreased in Ubc9 knockdown cells. The physphorylation of Akt showed similar trends. In addition, the inhibitor of PI3K/Akt signaling pathway, LY294002, dramatically inhibited the growth of Ubc9 overexpressing cells. Therefore, Ubc9 gene plays an important role in cell proliferation in EOC through PI3K/Akt signaling pathway. Full article

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 10⁶ pfu/mL and 1.56 × 10⁹ pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. Full article

Recently, senescence marker protein-30 (SMP30) knockout (KO) mice have been reported to be susceptible to apoptosis, however, the role of SMP30 has not been characterized in the small intestine. The aim of the present study is to investigate the role of SMP30 in the process of spontaneous and γ-radiation-induced apoptosis in mouse small intestine. Eight-week-old male wild-type (WT) mice and SMP30 KO mice were examined after exposure to 0, 1, 3, 5, and 9 Gy of γ-radiation. Apoptosis in the crypts of the small intestine increased in the 0 to 5 Gy radiated SMP30 KO and WT mice. Radiation-induced apoptosis and the BAX/Bcl-2 ratio in the SMP30 KO mice were significantly increased in comparison to each identically treated group of WT mice (p < 0.05). The levels of spontaneous apoptosis in both WT and KO mice were similar (p > 0.05), indicating that increased apoptosis of crypt cells of SMP30 KO by irradiation can be associated with SMP30 depletion. These results suggested that SMP30 might be involved in overriding the apoptotic homeostatic mechanism in response to DNA damage. Full article

Early exercise within 24 h after stroke can reduce neurological deficits after ischemic brain injury. However, the mechanisms underlying this neuroprotection remain poorly understood. Ischemic brain injury disrupts the blood-brain barrier (BBB) and then triggers a cascade of events, leading to secondary brain injury and poor long-term outcomes. This study verified the hypothesis that early exercise protected the BBB after ischemia. Adult rats were randomly assigned to sham, early exercise (EE) or non-exercise (NE) groups. The EE and NE groups were subjected to ischemia induced by middle cerebral artery occlusion (MCAO). The EE group ran on a treadmill beginning 24 h after ischemia, 30 min per day for three days. After three-days’ exercise, EB extravasation and electron microscopy were used to evaluate the integrity of the BBB. Neurological deficits, cerebral infarct volume and the expression of MMP-9, the tissue inhibitors of metalloproteinase-1 (TIMP-1), and occludin were determined. The data indicated that early exercise significantly inhibited the ischemia-induced reduction of occludin, and an increase in MMP-9 promoted TIMP-1 expression (p < 0.01), attenuated the BBB disruption (p < 0.05) and neurological deficits (p < 0.01) and diminished the infarct volume (p < 0.01). Our results suggest that the neuroprotection conferred by early exercise was likely achieved by improving the function of the BBB via the regulation of MMP-9 and occludin. Full article

In order to investigate the microbe-mineral interaction in the micro scale, spatial distribution and speciation of Cu and S in Halothiobacillus HT1 biofilm formed on a CuS surface was examined using synchrotron-based X-ray techniques. Confocal laser scanning microscope (CLSM) results indicated that Halothiobacillus HT1 biofilm formation gave rise to distinct chemical and redox gradients, leading to diverse niches in the biofilm. Live cells were distributed at the air-biofilm and membrane-biofilm interface. CuS was oxidized by Halothiobacillus HT1 biofilm, and copper penetrated into the biofilm. Sulfide was oxidized to cysteine (77.3%), sulfite (3.8%) and sulfonate (18.9%). Cu-cysteine-like species were involved in the copper homeostasis. These results significantly improve our understanding of the interfacial properties of the biofilm-mineral interface. Full article

Soil contamination by chromium (Cr) has become an increasing problem worldwide as a result of extensive industrial activities. Chromium, especially hexavalent Cr, impairs the growth and productivity of plants. Although it has been proposed that plants could modify their metabolism to adapt to Cr stress by reprogramming the expression of genes, especially those related to the antioxidant system, damage response, and electron transport chain, evidence at the protein expression level is lacking. To better understand the precise mechanisms underlying Cr phytoxicity and the plant response to Cr exposure, the time-course of changes in the protein expression profile induced by short-term hexavalent Cr exposure (1, 6 and 24 h) were analyzed in maize leaves. Among the over 1200 protein spots detected reproducibly by two-dimensional electrophoresis (2-DE), 60 were found to be differentially accumulated during Cr stress treatment. Of the Cr-regulated proteins, 58 were identified using tandem mass spectrometry (MS/MS). The Cr-regulated proteins identified were mainly involved in ROS detoxification and defense responses (26%), photosynthesis and chloroplast organization (22%), post-transcriptional processing of mRNA and rRNA (12%), protein synthesis and folding (10%), the DNA damage response (5%), and the cytoskeleton (3%). The possible involvement of these Cr stress-responsive proteins in Cr phytoxicity and the plant response to Cr exposure in maize is discussed, taking into consideration the information available from other plant models. Our results provide preliminary evidence that will facilitate understanding the molecular mechanisms underlying Cr toxicity in maize. Full article

The aim of this study was to investigate the prognostic value of tumor markers in operable non-small cell lung cancer (NSCLC) patients. A total of 481 NSCLC patients were enrolled in the present study. High levels of neuron-specific enolase (NSE), carbohydrate antigen 125 (CA125) and squamous cell carcinoma antigen (SCC) were detected in 306 (63.6%), 89 (18.5%) and 125 (26.0%) patients, respectively. Seventy-eight of 481 patients died of disease progression, and the median disease-free survival (DFS) and overall survival (OS) were 16.0 and 21.0 months, respectively. The three-year DFS rate was 56.7%, and the OS rate was 75.3%. For serum NSE, the three-year cumulative DFS rate for the normal and elevated group was 67.7% and 51.8% (p = 0.007). The OS in patients with high and normal levels of NSE was 34.0 months and 48.0 months, respectively. The median DFS was 46.0 months versus 32.0 months (p = 0.001), and the OS was 48.0 months versus 44.0 months (p = 0.001) in patients with normal and high levels of CA125. For patients with squamous cell carcinoma, the overall survival was significantly shorter in patients with elevated levels of SCC (p = 0.041). In the multivariate analysis high levels of NSE, CA125 and clinical stage were significantly correlated with worse prognosis (p < 0.05). Patients with all three tumor markers elevated presented the worst prognosis (p < 0.05). In our analysis, high levels of preoperative serum NSE and CA125 are correlated with worse survival in operable NSCLC patients. Full article

Hertwig’s epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro. Full article

Most of the intracellular endogenous microRNAs (endo-miRNAs) are considered to be saturated in Argonaute (Ago) proteins in the RNA-induced silencing complexes (RISCs). When exogenous miRNAs (exo-miRNAs) are introduced into cells, endo-miRNAs in the RISC may be replaced with exo-miRNAs or exo-miRNAs, and endo-miRNAs might also compete for the position in the newly synthesized RISC with each other. This would lead to the fluctuation of global gene expression not only by repression of exo-miRNA target gene expression, but also by the increase of the endo-miRNA target gene expression. In the present study, we quantified the changes in the expression levels of target genes of exo-miRNA and endo-miRNA in the cells transfected with fifteen different exo-miRNAs by microarray experiments. Different exo-miRNAs increased ratios of expression levels of target genes of a given endo-miRNA to different extents, suggesting that the replacement efficiencies might differ according to the exo-miRNA types. However, the increased ratios in the expression levels of each endo-miRNA target genes by the transfection of any particular exo-miRNA were mostly equivalent, suggesting that the endo-miRNAs present in the RISC might be replaced with excessive exo-miRNAs at similar levels, probably because they exist in single-stranded forms in the RISC. Full article

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, miRNAs involved in hypertension have not been thoroughly investigated. We have recently reported that specific miRNAs play an integral role in Angiotensin II receptor (AT₁R) signaling, especially after activation of the Gαq signaling pathway. Since AT₁R blockers are widely used to treat hypertension, we undertook a detailed analysis of potential miRNAs involved in Angiotensin II (AngII) mediated hypertension in rats and hypertensive patients, using miRNA microarray and qPCR analysis. The miR-132 and miR-212 are highly increased in the heart, aortic wall and kidney of rats with hypertension (159 ± 12 mm Hg) and cardiac hypertrophy following chronic AngII infusion. In addition, activation of the endothelin receptor, another Gαq coupled receptor, also increased miR-132 and miR-212. We sought to extend these observations using human samples by reasoning that AT₁R blockers may decrease miR-132 and miR-212. We analyzed tissue samples of mammary artery obtained from surplus arterial tissue after coronary bypass operations. Indeed, we found a decrease in expression levels of miR-132 and miR-212 in human arteries from bypass-operated patients treated with AT₁R blockers, whereas treatment with β-blockers had no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms. Full article

Cisplatin is one of the most widely used chemical drugs for anticancer treatment. Recent studies have focused on the ability of cisplatin to retain the high mobility group box 1 (HMGB1) protein in cisplatin-DNA adducts, thereby preventing its release from the nucleus. Because HMGB1 is a powerful inflammatory mediator in many diseases, the aim of this study is to evaluate the therapeutic effect of cisplatin acute liver failure. In this study, low-dose cisplatin was administered to treat PMA-induced macrophage-like cells induced by PMA and rats with acute liver failure. We found that cell viability and liver injury were greatly improved by cisplatin treatment. The extracellular levels of HMGB1, TNF-α and IFN-γ were also significantly decreased by the administration of cisplatin. During inflammation, nuclear HMGB1 translocates from the nucleus to the cytoplasm. The administration of cisplatin reduced the cytoplasmic levels of HMGB1 and increased nuclear HMGB1 levels in vitro and in vivo. In conclusion, cisplatin can protect against acute liver failure by retaining HMGB1 in the nucleus and preventing its release into the extracellular milieu. Full article

Extending our previous observations, we have shown on HaCat cells that melatonin, at ~10⁻⁹ M concentration, transiently raises not only the expression of the neuronal nitric oxide synthase (nNOS) mRNA, but also the nNOS protein synthesis and the nitric oxide oxidation products, nitrite and nitrate. Interestingly, from the cell bioenergetic point of view, the activated NO-related chemistry induces a mild decrease of the oxidative phosphorylation (OXPHOS) efficiency, paralleled by a depression of the mitochondrial membrane potential. The OXPHOS depression is apparently balanced by glycolysis. The mitochondrial effects described have been detected only at nanomolar concentration of melatonin and within a time window of a few hours’ incubation; both findings compatible with the melatonin circadian cycle. Full article

An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring. Full article

PCA3 (prostate cancer gene 3) and multiparametric 3 tesla MRI are new promising diagnostic tools in the detection of PCa. Our aim was to study the clinical value of the Progensa PCA3-test: its predictive value for biopsy outcome, Gleason score and MRI outcome. We evaluated, retrospectively, 591 patients who underwent a Progensa PCA3-test at the Radboud University Nijmegen Medical Centre between May 2006 and December 2009. Prostate biopsies were performed in 290 patients; a multiparametric 3 tesla MRI of the prostate was performed in 163/591 patients. The PCA3-score was correlated to biopsy results and MRI outcome. The results show that PCA3 was highly predictive for biopsy outcome (p < 0.001); there was no correlation with the Gleason score upon biopsy (p = 0.194). The PCA3-score of patients with a suspicious region for PCa on MRI was significantly higher (p < 0.001) than in patients with no suspicious region on MRI (52 vs. 21). In conclusion, PCA3 is a valuable diagnostic biomarker for PCa; it did not correlate with the Gleason score. Furthermore, multiparametric MRI outcome was significantly correlated with the PCA3-score. Thus, PCA3 could be used to select patients that require MRI. However, in patients with a negative PCA3 and high clinical suspicion of PCa, a multiparametric MRI should also be done. Full article

Pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is the most destructive diseases of pine and poses a threat of serious economic losses worldwide. Although several of the mechanisms involved in disease progression have been discovered, the molecular response of Pinus massoniana to PWN infection has not been explored. We constructed four subtractive suppression hybridization cDNA libraries by taking time-course samples from PWN-inoculated Masson pine trees. One-hundred forty-four significantly differentially expressed sequence tags (ESTs) were identified, and 124 high-quality sequences with transcriptional features were selected for gene ontology (GO) and individual gene analyses. There were marked differences in the types of transcripts, as well as in the timing and levels of transcript expression in the pine trees following PWN inoculation. Genes involved in signal transduction, transcription and translation and secondary metabolism were highly expressed after 24 h and 72 h, while stress response genes were highly expressed only after 72 h. Certain transcripts responding to PWN infection were discriminative; pathogenesis and cell wall-related genes were more abundant, while detoxification or redox process-related genes were less abundant. This study provides new insights into the molecular mechanisms that control the biochemical and physiological responses of pine trees to PWN infection, particularly during the initial stage of infection. Full article

RNA polymerase type I (plastid-encoded polymerase, PEP) is one of the key chloroplast enzymes. However, the rpo genes that encode its subunits (rpoA, rpoB, rpoC1 and rpoC2) are relatively rapidly evolving sequences. The aim of this study was to investigate the rate of the molecular evolution of rpo genes and to evaluate them as phylogenetic markers on the example of the genus Lamium L. (Lamiaceae). The analyzed genes were shown to differ in the level of variation, rate of intragenic mutations, phylogenetic informativeness, and in the impact of these mutations on the properties of encoded peptides. Destabilizing effects of the positive pressure were observed in all genes examined coding for PEP enzyme. We have demonstrated the relationship between mutations fixed by positive selection and the separation of phylogenetic lines within the genus Lamium. The study showed also that the rpo genes were reliable phylogenetic markers, useful in the reconstruction of interconnections of species belonging to the same genus. Of the four tested genes, the most promising phylogenetic marker was rpoA gene, while the least useful gene appeared to be rpoC1. Full article

The -173 G/C polymorphism in the macrophage migration inhibitory factor (MIF) gene has been implicated in susceptibility to inflammatory bowel disease (IBD), but the results are inconclusive. The present meta-analysis aimed to investigate the overall association between the -173 G/C polymorphism and IBD risk. We searched in Pubmed, and Embase for studies evaluating the association between the -173G/C gene polymorphism and IBD risk. Data were extracted and statistical analysis was performed using Revman 5.1 and STATA 12.0 software. A total of seven publications involving 4729 subjects (2282 IBD cases and 2447 controls) were included in this meta-analysis. Combined analysis revealed a clear association between this polymorphism and IBD susceptibility (OR = 1.48, 95% CI: 1.10–2.00, p = 0.009 for CC vs. CG + GG). Subgroup analysis by ethnicity showed that the IBD risk associated with the -173G/C gene polymorphism was significantly elevated among Asians (OR = 1.79, 95% CI: 1.08–2.96, p = 0.02), but not among Caucasians. Subgroup analysis by disease suggested that the -173G/C gene polymorphism is a risk factor for ulcerative colitis (OR = 1.62, 95% CI: 1.10–2.37, p = 0.01), but that it was not associated with Crohn’s disease. This meta-analysis suggests that the -173 G/C polymorphism in the macrophage MIF gene contributes to IBD susceptibility, specifically in Asian populations. Further studies are needed to validate these findings. Full article

Spinal muscular atrophy (SMA) is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN) protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD). In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons. Full article

Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep. Full article

In this work, Fe₃O₄@SiO₂ nanoparticles were coated with mesoporous silica shell by S⁻N⁺I⁻ pathway by using anionic surfactant (S⁻) and co-structure directing agent (N⁺). The role of co-structure directing agent (CSDA) is to assist the electrostatic interaction between negatively charged silica layers and the negatively charged surfactant molecules. Prior to the mesoporous shell formation step, magnetic cores were coated with a dense silica layer to prevent iron oxide cores from leaching into the mother system under any acidic circumstances. However, it was found that both dense and mesoporous coating parameters affect the textural properties of the produced mesoporous silica shell (i.e., surface area, pore volume and shell thickness). The synthesized Fe₃O₄@SiO₂@m-SiO₂ (MCMSS) nanoparticles have been characterized by low-angle X-ray diffraction, transmission electron microscopy (TEM), and N₂ adsorption-desorption analysis, and magnetic properties. The synthesized particles had dense and mesoporous silica shells of 8–37 nm and 26–50 nm, respectively. Furthermore, MCMSS possessed surface area of ca. 259–621 m²·g⁻¹, and pore volume of ca. 0.216–0.443 cc·g⁻¹. MCMSS showed docetaxcel cancer drug storage capacity of 25–33 w/w% and possessed control release from their mesochannels which suggest them as proper nanocarriers for docetaxcel molecules. Full article

Molecular Dynamics is a method of choice for membrane simulations and the rising of coarse-grained forcefields has opened the way to longer simulations with reduced calculations times. Here, we present an elastic network, SAHBNET (Surface Accessibility Hydrogen-Bonds elastic NETwork), that will maintain the structure of soluble or membrane proteins based on the hydrogen bonds present in the atomistic structure and the proximity between buried residues. This network is applied on the coarse-grained beads defined by the MARTINI model, and was designed to be more physics-based than a simple elastic network. The SAHBNET model is evaluated against atomistic simulations, and compared with ELNEDYN models. The SAHBNET is then used to simulate two membrane proteins inserted in complex lipid bilayers. These bilayers are formed by self-assembly and the use of a modified version of the GROMACS tool genbox (which is accessible through the gcgs.gembloux.ulg.ac.be website). The results show that SAHBNET keeps the structure close to the atomistic one and is successfully used for the simulation of membrane proteins. Full article

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization. Full article

MicroRNAs, which are small endogenous RNA regulators, have been associated with various types of cancer. Breast cancer is a major health threat for women worldwide. Many miRNAs were reported to be associated with the progression and carcinogenesis of breast cancer. In this study, we aimed to discover novel breast cancer-related miRNAs and to elucidate their functions. First, we identified confident miRNA-target pairs by combining data from miRNA target prediction databases and expression profiles of miRNA and mRNA. Then, miRNA-regulated protein interaction networks (PINs) were constructed with confident pairs and known interaction data in the human protein reference database (HPRD). Finally, the functions of miRNA-regulated PINs were elucidated by functional enrichment analysis. From the results, we identified some previously reported breast cancer-related miRNAs and functions of the PINs, e.g., miR-125b, miR-125a, miR-21, and miR-497. Some novel miRNAs without known association to breast cancer were also found, and the putative functions of their PINs were also elucidated. These include miR-139 and miR-383. Furthermore, we validated our results by receiver operating characteristic (ROC) curve analysis using our miRNA expression profile data, gene expression-based outcome for breast cancer online (GOBO) survival analysis, and a literature search. Our results may provide new insights for research in breast cancer-associated miRNAs. Full article

Glucosinolates, a class of secondary metabolites, mainly found in Brassicaceae, are affected by the changing environment. This review is focusing on the physiological significance of glucosinolates and their hydrolysis products in the plant response to different abiotic stresses. Special attention is paid to the crosstalk between some of the physiological processes involved in stress response and glucosinolate metabolism, with the resulting connection between both pathways in which signaling mechanisms glucosinolate may act as signals themselves. The function of glucosinolates, further than in defense switching, is discussed in terms of alleviating pathogen attack under abiotic stress. The fact that the exogenous addition of glucosinolate hydrolysis products may alleviate certain stress conditions through its effect on specific proteins is described in light of the recent reports, but the molecular mechanisms involved in this response merit further research. Finally, the transient allocation and re-distribution of glucosinolates as a response to environmental changes is summarized. Full article

The photooxidation of cellular monounsaturated fatty acids was investigated in senescent phytoplanktonic cells (Emiliania huxleyi) and in their attached bacteria under laboratory controlled conditions. Our results indicated that UV-visible irradiation of phytodetritus induced the photooxidation of oleic (produced by phytoplankton and bacteria) and cis-vaccenic (specifically produced by bacteria) acids. These experiments confirmed the involvement of a substantial singlet oxygen transfer from senescent phytoplanktonic cells to attached bacteria, and revealed a significant correlation between the concentration of chlorophyll, a photosensitizer, in the phytodetritus and the photodegradation state of bacteria. Hydroperoxyacids (fatty acid photoproducts) appeared to be quickly degraded to ketoacids and hydroxyacids in bacteria and in phytoplanktonic cells. This degradation involves homolytic cleavage (most likely induced by UV and/or transition metal ions) and peroxygenase activity (yielding epoxy acids). Full article

Prostate cancer is widely observed to be biologically heterogeneous. Its heterogeneity is manifested histologically as multifocal prostate cancer, which is observed more frequently than unifocal prostate cancer. The clinical and prognostic significance of either focal cancer type is not fully established. To investigate prostate cancer heterogeneity, the genetic profiles of multifocal and unifocal prostate cancers were compared. Here, we report observations deduced from tumor-tumor comparison of copy number alteration data of both focal categories. Forty-one fresh frozen prostate cancer foci from 14 multifocal prostate cancers and eight unifocal prostate cancers were subjected to copy number variation analysis with the Affymetrix SNP 6.0 microarray tool. With the investigated cases, tumors obtained from a single prostate exhibited different genetic profiles of variable degrees. Further comparison identified no distinct genetic pattern or signatures specific to multifocal or unifocal prostate cancer. Our findings suggest that samples obtained from multiple sites of a single unifocal prostate cancer show as much genetic heterogeneity and variability as separate tumors obtained from a single multifocal prostate cancer. Full article

Some antioxidants have been shown to possess additional pro-oxidant effects. Diverse methodologies exist for studying redox properties of synthetic and natural chemicals. The latter are substantial components of our diet. Exploration of their contribution to life-extending or -compromising effects is mandatory. Among reactive oxygen species (ROS), hydroxyl radical (^•OH) is the most damaging species. Due to its short half-life, the assay has to contain a specific generation system. Plants synthesize flavonoids, phenolic compounds recognized as counter-agents to coronary heart disease. Their antioxidant activities are affected by their hydroxylation patterns. Moreover, in the plant, they mainly occur as glycosides. We chose three derivatives, quercetin, luteolin, and rutin, in attempts to explore their redox chemistry in contrasting hydrogen peroxide environments. Initial addition of hydrogen peroxide in high concentration or gradual development constituted a main factor affecting their redox chemical properties, especially in case of quercetin. Our study exemplifies that a combination of a chemical assay (deoxyribose degradation) with an electrochemical method (square-wave voltammetry) provides insightful data. The ambiguity of the tested flavonoids to act either as anti- or pro-oxidant may complicate categorization, but probably contributed to their evolution as components of a successful metabolic system that benefits both producer and consumer. Full article

We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. Full article

The aim of this study was to compare the anti-adhesion efficacy of a bi-layer electrospun fibrous membrane consisting of hyaluronic acid-loaded poly(ε-caprolactone) (PCL) fibrous membrane as the inner layer and PCL fibrous membrane as the outer layer with a single-layer PCL electrospun fibrous membrane in a rat cecum abrasion model. The rat model utilized a cecal abrasion and abdominal wall insult surgical protocol. The bi-layer and PCL membranes were applied between the cecum and the abdominal wall, respectively. Control animals did not receive any treatment. After postoperative day 14, a visual semiquantitative grading scale was used to grade the extent of adhesion. Histological analysis was performed to reveal the features of adhesion tissues. Bi-layer membrane treated animals showed significantly lower adhesion scores than control animals (p < 0.05) and a lower adhesion score compared with the PCL membrane. Histological analysis of the bi-layer membrane treated rat rarely demonstrated tissue adhesion while that of the PCL membrane treated rat and control rat showed loose and dense adhesion tissues, respectively. Bi-layer membrane can efficiently prevent adhesion formation in abdominal cavity and showed a significantly decreased adhesion tissue formation compared with the control. Full article

Cabbage is a relatively robust vegetable at low temperatures. However, at high temperatures, cabbage has disadvantages, such as reduced disease tolerance and lower yields. Thus, selection of heat-tolerant cabbage is an important goal in cabbage breeding. Easier or faster selection of superior varieties of cabbage, which are tolerant to heat and disease and have improved taste and quality, can be achieved with molecular and biological methods. We compared heat-responsive gene expression between a heat-tolerant cabbage line (HTCL), “HO”, and a heat-sensitive cabbage line (HSCL), “JK”, by Genechip assay. Expression levels of specific heat stress-related genes were increased in response to high-temperature stress, according to Genechip assays. We performed quantitative RT-PCR (qRT-PCR) to compare expression levels of these heat stress-related genes in four HTCLs and four HSCLs. Transcript levels for heat shock protein BoHsp70 and transcription factor BoGRAS (SCL13) were more strongly expressed only in all HTCLs compared to all HSCLs, showing much lower level expressions at the young plant stage under heat stress (HS). Thus, we suggest that expression levels of these genes may be early selection markers for HTCLs in cabbage breeding. In addition, several genes that are involved in the secondary metabolite pathway were differentially regulated in HTCL and HSCL exposed to heat stress. Full article

Myricetin is a naturally occurring flavonol found in many plant based food sources. It increases the lifespan of Caenorhabditis elegans, but the molecular mechanisms are not yet fully understood. We have investigated the impact of this flavonoid on the transcription factors DAF-16 (C. elegans FoxO homologue) and SKN-1 (Nrf2 homologue), which have crucial functions in the regulation of ageing. Myricetin is rapidly assimilated by the nematode, causes a nuclear translocation of DAF-16 but not of SKN-1, and finally prolongs the mean adult lifespan of C. elegans by 32.9%. The lifespan prolongation was associated with a decrease in the accumulation of reactive oxygen species (ROS) detected by DCF. Myricetin also decreases the formation of lipofuscin, a pigment consisting of highly oxidized and cross-linked proteins that is considered as a biomarker of ageing in diverse species. The lifespan extension was completely abolished in a daf-16 loss-of-function mutant strain (CF1038). Consistently with this result, myricetin was also not able to diminish stress-induced ROS accumulation in the mutant. These results strongly indicate that the pro-longevity effect of myricetin is dependent on DAF-16 and not on direct anti-oxidative effects of the flavonoid. Full article

1,5-Dimethyl-4-((2-methylbenzylidene)amino)-2-phenyl-1H-pyrazol-3(2H)-one (DMPO) was synthesized to be evaluated as a corrosion inhibitor. The corrosion inhibitory effects of DMPO on mild steel in 1.0 M HCl were investigated using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, open circuit potential (OCP) and electrochemical frequency modulation (EFM). The results showed that DMPO inhibited mild steel corrosion in acid solution and indicated that the inhibition efficiency increased with increasing inhibitor concentration. Changes in the impedance parameters suggested an adsorption of DMPO onto the mild steel surface, leading to the formation of protective films. The novel synthesized corrosion inhibitor was characterized using UV-Vis, FT-IR and NMR spectral analyses. Electronic properties such as highest occupied molecular orbital energy, lowest unoccupied molecular orbital energy (EHOMO and ELUMO, respectively) and dipole moment (μ) were calculated and discussed. The results showed that the corrosion inhibition efficiency increased with an increase in the EHOMO values but with a decrease in the ELUMO value. Full article

Excessive ethanol consumption, obesity and increasing age may all lead to increased serum levels of gamma-glutamyltransferase (GGT) enzyme, which plays a key role in the metabolism of extracellular reduced glutathione. However, as yet, the interactions between the various modulators of GGT activities have remained poorly defined. We analyzed data from 15,617 apparently healthy individuals (7254 men and 8363 women, mean age 46 ± 13 years, range 25–74 years) who participated in a national cross-sectional health survey in Finland between 1997 and 2007. All subjects underwent detailed clinical examinations and interviews, including the amount of ethanol use and smoking habits. GGT levels were measured from all participants, and the individual and joint impacts of the different study variables on GGT levels were assessed. Significant individual effects were noted for ethanol use (p < 0.001), body mass index (BMI) (p < 0.001), age (p < 0.001) and smoking (p < 0.001). In men, significant two-factor interactions occurred between ethanol use and age (p < 0.020). Among those over 40 years of age, ethanol consumption was found to be a stronger determinant of increased GGT levels than in men below 40 years, whereas in the latter age group, BMI was found to predominate. In women, a significant two-factor interaction occurred between ethanol and BMI (p = 0.010), whereas it did not with ethanol use and age. The data underscores the role of ethanol consumption and age as major determinants of increased GGT levels in men, whereas in women, a relatively stronger impact was noted for ethanol intake and BMI. In light of the ability of GGT enzyme to modulate crucial redox-sensitive functions, the present findings also support the use of GGT as a biomarker of oxidative stress. Full article

Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysis revealed that both ODNs were effective in reducing their target proteins when administered alone or in combination. In addition, treatment with the ODNs was associated with an induction of apoptosis. Our data suggest that dual targeting of the AR and PKARIα is more effective in inhibiting LNCaP and LNCaPabl tumor growth than single treatment and may give a treatment benefit, especially in castration-resistant prostate cancers. Full article

Enhancer of zeste homolog 2 (EZH2) plays a crucial role in embryonic and somatic stem cells for their proliferation and differentiation. However, the roles and underlying mechanisms of EZH2 in prostate cancer stem cells (PCSCs) remain unknown. This study aimed to investigate the effects of EZH2 on PCSCs. PCSCs were isolated from the human prostate cancer cell line LNcap by fluorescence activated cell sorting (FACS). EZH2 expression was compared between PCSCs and non-PCSCs. The association between EZH2 function and PCSC growth was investigated using siRNA-mediated knock-down of EZH2. Cell growth was investigated by MTT, cell cycle and apoptosis of PCSCs were explored by flow cytometric analysis. Finally, the upstream pathway miRNA level was determined via a luciferase reporter assay, and the downstream pathway cycle regulators were examined via reverse transcriptase-polymerase chain reaction. The results showed that LNcap cell line comprised a greater proportion of CD44⁺/CD133⁺ cells by comparison to the PC-3 cell line. EZH2 was up-regulated in PCSCs compared with non-PCSCs. Silence of EZH2 inhibited cell growth and the cell cycle and promoted the progression of apoptosis. Furthermore, EZH2 was a direct target of miR-101 in PCSCs and EZH2’s mRNA levels were inversely correlated with miR-101 expression and cyclin E2 (a cell-cycle regulator) was suppressed by siEZH2. In conclusion, EZH2 is essential for PCSC growth, partly through a negative regulation by miR-101 and positively regulating cyclin E2. Full article

The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD⁺ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak. Full article

Certain anaerobic bacterial species tend to predominate the vaginal flora during bacterial vaginosis (BV), with Gardnerella vaginalis being the most common. However, the exact role of G. vaginalis in BV has not yet been determined. The main goal of this study was to test the hypothesis that G. vaginalis is an early colonizer, paving the way for intermediate (e.g., Fusobacterium nucleatum) and late colonizers (e.g., Prevotella bivia). Theoretically, in order to function as an early colonizer, species would need to be able to adhere to vaginal epithelium, even in the presence of vaginal lactobacilli. Therefore, we quantified adherence of G. vaginalis and other BV-associated bacteria to an inert surface pre-coated with Lactobacillus crispatus using a new Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) methodology. We found that G. vaginalis had the greatest capacity to adhere in the presence of L. crispatus. Theoretically, an early colonizer would contribute to the adherence and/or growth of additional species, so we next quantified the effect of G. vaginalis biofilms on the adherence and growth of other BV-associated species by quantitative Polymerase Chain Reaction (qPCR) technique. Interestingly, G. vaginalis derived a growth benefit from the addition of a second species, regardless of the species. Conversely, G. vaginalis biofilms enhanced the growth of P. bivia, and to a minor extent of F. nucleatum. These results contribute to our understanding of BV biofilm formation and the progression of the disorder. Full article

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a dual-specificity lipid and protein phosphatase. The loss of PTEN was originally discovered in numerous human cancers. PTEN inhibition by bisperoxovanadium (bpV) reduces neurological damage after ischemic brain injury. The purpose of this study was to identify the optimal neuroprotective dose of bpV when administrated after focal ischemia/reperfusion (I/R) injury in rats. Focal I/R injury was induced using the middle cerebral artery occlusion method. bpV at doses of 0.25, 0.50 and 1.0 mg/kg were injected intraperitoneally just after reperfusion, with saline serving as a vehicle control. A maximal reduction in brain injury was observed with 1.0 mg/kg bpV. This dose of bpV also significantly blocked apoptosis in the penumbral cortex of rats. This beneficial effect was associated with the increasing levels of Akt phosphorylation in the penumbral cortex. These results demonstrate that the pharmacological inhibition of PTEN protects against I/R injury in a dose-dependent manner and the protective effect might be induced through upregulation of the phosphoinositide-3 kinase/Akt pro-survival pathway, suggesting a new therapeutic strategy to combat ischemic brain injury. Full article

(−)-Epigallocatechin gallate (EGCG) and curcumin are two naturally derived agents that have been widely investigated worldwide. They exhibit their anti-tumor effects in many types of cancers. In the current study, the effect of the combination of the two agents on non-small cell lung cancer (NSCLC) cells was investigated. The results revealed that at low concentrations, the combination of the EGCG and curcumin strongly enhanced cell cycle arrest. Flow cytometry analysis showed that the cells were arrested at G1 and S/G2 phases. Two main cell cycle related proteins cyclin D1 and cyclin B1 were significantly inhibited at the present of EGCG and curcumin. EdU (5-ethynyl-2'-deoxyuridine) fluorescence staining showed that the DNA replication was significantly blocked. A clonal growth assay also confirmed a marked repression of cell growth. In a lung cancer xenograft node mice model, combination of EGCG and curcumin exhibited protective effect against weight loss due to tumor burden. Tumor growth was strongly repressed by the combination of the two agents, without causing any serious side-effect. Overall, these results strongly suggest that EGCG in combination with curcumin could be a candidate for chemoprevention agent of NSCLC. Full article

Janus kinase 2 (JAK2) is an intracellular nonreceptor tyrosine kinase that belongs to the JAK family of kinases, which play an important role in survival, proliferation, and differentiation of a variety of cells. JAK2 inhibitors are potential drugs for the treatment of myeloproliferative neoplasms. The three dimensional quantitative structure-activity relationships have been studied on a series of JAK2 inhibitors by comparative molecular field analysis (CoMFA), and comparative molecular similarity indices analysis (CoMSIA). The CoMFA model had a cross-validated coefficient q² of 0.633, and the relation non-cross-validated coefficient r² of 0.976. The F value is 225.030. The contributions of steric and electrostatic fields to the activity are 55.2% and 44.8%, respectively. For the CoMSIA study, the q², r², and F values of the model are 0.614, 0.929, and 88.771, respectively. The contributions of steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond donor fields to the activity are 27.3%, 23.9%, 16.4%, 21.7%, and 10.7%, respectively. The CoMFA and CoMSIA models showed strong predictive ability, and the 3D contour plots give the basis on the structure modification of JAK2 inhibitors. Full article

Potent drugs are desperately needed to counteract bacterial biofilm infections, especially those caused by gram-positive organisms, such as Staphylococcus aureus. Moreover, anti-biofilm compounds/agents that can be used as chemical tools are also needed for basic in vitro or in vivo studies aimed at exploring biofilms behavior and functionability. In this contribution, a collection of naturally-occurring abietane-type diterpenes and their derivatives was tested against S. aureus biofilms using a platform consisting of two phenotypic assays that have been previously published by our group. Three active compounds were identified: nordehydroabietylamine (1), (+)-dehydroabietic acid (2) and (+)-dehydroabietylamine (3) that prevented biofilm formation in the low micromolar range, and unlike typical antibiotics, only 2 to 4-fold higher concentrations were needed to significantly reduce viability and biomass of existing biofilms. Compound 2, (+)-dehydroabietic acid, was the most selective towards biofilm bacteria, achieving high killing efficacy (based on log Reduction values) and it was best tolerated by three different mammalian cell lines. Since (+)-dehydroabietic acid is an easily available compound, it holds great potential to be used as a molecular probe in biofilms-related studies as well as to serve as inspirational chemical model for the development of potent drug candidates. Full article

Poly(hydroxyethyl methacrylate-co-glycidyl methacrylate)-grafted magnetic chitosan microspheres (HG-MCM) were prepared using reversed-phase suspension polymerization method. The HG-MCM presented a core-shell structure and regular spherical shape with poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) grafted onto the chitosan layer coating the Fe₃O₄ cores. The average diameter of the magnetic microspheres was 10.67 μm, within a narrow size distribution of 6.6–17.4 μm. The saturation magnetization and retentivity of the magnetic microspheres were 7.0033 emu/g and 0.6273 emu/g, respectively. The application of HG-MCM in immobilization of lactase showed that the immobilized enzyme presented higher storage, pH and thermal stability compared to the free enzyme. This indicates that HG-MCM have potential applications in bio-macromolecule immobilization. Full article

Eviprostat is a popular phytotherapeutic agent for the treatment of lower urinary tract symptoms (LUTS). At present, the signaling mechanisms underlying its therapeutic effects are still poorly understood. Given that cAMP has been reported to suppress cell hyperplasia and hypertrophy in various pathological situations, we asked whether the effect of Eviprostat could be ascribed to the activation of the cAMP signaling pathway. In the study, exposure of cAMP response element (CRE)-secreted alkaline phosphatase (SEAP) (CRE-SEAP)-reporter cells to Eviprostat elevated SEAP secretion, which was associated with an increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and cAMP-response element-binding protein (CREB), as well as enhanced expression of CRE-regulated protein connexin43, indicating an activation of the cAMP signaling pathway. Consistent with these observations, Eviprostat-induced expression of Cx43 was abolished in the presence of adenylyl cyclase inhibitor SQ22536 or PKA inhibitor H89, whereas it was mimicked by adenylyl cyclase activator, forskolin. Further analysis demonstrated that Eviprostat significantly potentiated the effect of phosphodiesterase 3 (PDE3) inhibitor, but not that of PDE4 inhibitor, on CRE activation. Moreover, Eviprostat suppressed PDGF-induced activation of ERK and Akt and inhibited cell proliferation and hillock formation in both mesangial cells and bladder smooth muscle cells. Collectively, activation of the cAMP signaling pathway could be an important mechanism by which Eviprostat exerts its therapeutic effects for LUTS. Full article

Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction. Full article

Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, the compact energetic map of the native state is generated by a preliminary short molecular dynamics (MD) simulation of the protein in explicit solvent. This map is simplified by means of an eigenvalue decomposition. The highest components of the eigenvector associated with the lowest eigenvalue indicate which sites, named “hot spots”, are likely to be responsible for the stability and correct folding of the protein. In the Hamiltonian replica exchange protocol, we use modified force-field parameters to treat the interparticle non-bonded potentials of the hot spots within the protein and between protein and solvent atoms, leaving unperturbed those relative to all other residues, as well as solvent-solvent interactions. We show that it is possible to reversibly simulate the folding/unfolding behavior of two test proteins, namely Villin HeadPiece HP35 (35 residues) and Protein A (62 residues), using a limited number of replicas. We next discuss possible implications for the study of folding mechanisms via all atom simulations. Full article

Metallothioneins (MT) are low molecular weight, cysteine-rich proteins maintaining metal ions homeostasis. They play a role in carcinogenesis and may also cause chemoresistance. The aim of the study was to explore the importance of MT serum levels in children suffering from malignant tumours. This prospective study involves examination of 865 samples from 172 patients with malignant tumours treated from 2008 to 2011 at University Hospital Motol. MT serum levels were determined using differential pulse voltammetry–Brdicka reaction. Mean MT level was 2.7 ± 0.5 μM. There was no statistically significant difference between MT levels in different tumours. We also did not find any correlation between MT levels and response to therapy or clinical stages. However, we found a positive correlation between MT levels and age (p = 0.009) and a negative correlation with absolute lymphocyte number (p = 0.001). The fact that patients who had early disease recurrence had lower MT levels during the treatment (complete remission 2.67 vs. recurring 2.34, p = 0.001) seems to be important for clinical practice. Accordingly we believe that there is benefit in further studies of serum MT levels in tumours. Full article

Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10⁻⁵ M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABA_A receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains. Full article

In this study, the immuno-modulatory and anticancer activities of marine algae, Spirulina maxima grown in deep-sea water (DSW), were investigated. It was found that the extract of S. maxima, cultured in DSW, effectively suppressed the expression of Bcl2 in A549 cells as well as inhibiting various human cancer cells with concentration dependency, which possibly implies that the extracts may play more important roles in controlling cancer cell growth. The secretion of cytokines IL-6 and TNF-α from human B cells was also greatly increased, compared to those of the extract grown in conventional sea-water. The growth of Human Natural Killer (NK) cells in the presence of the extracts from DSW was significantly higher (12.2 × 10⁴ viable cells/mL) when compared to the control (1.1 × 10⁴ viable cells/mL). Based on HPLC analysis, the increase in the biological activities of the extracts from DSW was caused by considerably high amounts of β-carotene and ascorbic acid because the DSW contained high concentrations and good ratios of several key minerals for biosynthesizing β-carotene and ascorbic acid, as well as maintaining high cell growth. Full article

The human polycistronic miRNA cluster miR-17-92 is frequently overexpressed in hematopoietic malignancies and cancers. Its transcription is in part controlled by an E2F-regulated host gene promoter. An intronic A/T-rich region directly upstream of the miRNA coding region also contributes to cluster expression. Our deletion analysis of the A/T-rich region revealed a strong dependence on c-Myc binding to the functional E3 site. Yet, constructs lacking the 5'-proximal ~1.3 kb or 3'-distal ~0.1 kb of the 1.5 kb A/T-rich region still retained residual specific promoter activity, suggesting multiple transcription start sites (TSS) in this region. Furthermore, the protooncogenic kinase, Pim-1, its phosphorylation target HP1γ and c-Myc colocalize to the E3 region, as inferred from chromatin immunoprecipitation. Analysis of pri-miR-17-92 expression levels in K562 and HeLa cells revealed that silencing of E2F3, c-Myc or Pim-1 negatively affects cluster expression, with a synergistic effect caused by c-Myc/Pim-1 double knockdown in HeLa cells. Thus, we show, for the first time, that the protooncogene Pim-1 is part of the network that regulates transcription of the human miR-17-92 cluster. Full article

The overexpression of antiapoptotic genes, such as Bcl-xL and survivin, contributes to the increased survival of tumor cells and to the development of treatment resistances. In the bladder cancer cell lines EJ28 and J82, the siRNA-mediated knockdown of survivin reduces cell proliferation and the inhibition of Bcl-xL sensitizes these cells towards subsequent chemotherapy with mitomycin C and cisplatin. Therefore, the aim of this study was to analyze if the simultaneous knockdown of Bcl-xL and survivin might represent a more powerful treatment option for bladder cancer than the single inhibition of one of these target genes. At 96 h after transfection, reduction in cell viability was stronger after simultaneous inhibition of Bcl-xL and survivin (decrease of 40%–48%) in comparison to the single target treatments (decrease of 29% at best). Furthermore, simultaneous knockdown of Bcl-xL and survivin considerably increased the efficacy of subsequent chemotherapy. For example, cellular viability of EJ28 cells decreased to 6% in consequence of Bcl-xL and survivin inhibition plus cisplatin treatment whereas single target siRNA plus chemotherapy treatments mediated reductions down to 15%–36% only. In conclusion, the combination of simultaneous siRNA-mediated knockdown of antiapoptotic Bcl-xL and survivin—a multitarget molecular-based therapy—and conventional chemotherapy shows great potential for improving bladder cancer treatment. Full article

Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (K_D) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from −17.2 kcal/mol to −10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface. Full article

Most protein crystallisation begins from heterogeneous nucleation; in practice, crystallisation typically occurs in the presence of a solid surface in the solution. The solid surface provides a nucleation site such that the energy barrier for nucleation is lower on the surface than in the bulk solution. Different types of solid surfaces exhibit different surface energies, and the nucleation barriers depend on the characteristics of the solid surfaces. Therefore, treatment of the solid surface may alter the surface properties to increase the chance to obtain protein crystals. In this paper, we propose a method to modify the glass cover slip using a self-assembled monolayer (SAM) of functional groups (methyl, sulfydryl and amino), and we investigated the effect of each SAM on protein crystallisation. The results indicated that both crystallisation success rate in a reproducibility study, and crystallisation hits in a crystallisation screening study, were increased using the SAMs, among which, the methyl-modified SAM demonstrated the most significant improvement. These results illustrated that directly modifying the crystallisation plates or glass cover slips to create surfaces that favour heterogeneous nucleation can be potentially useful in practical protein crystallisation, and the utilisation of a SAM containing a functional group can be considered a promising technique for the treatment of the surfaces that will directly contact the crystallisation solution. Full article

The orphan GPR87 has recently been matched with its ligand LPA, which is a lipid mediator with multiple physiological functions, including cancer cell proliferation. This study aimed to clarify the role of GPR87 in urothelial carcinoma of the bladder. GPR87 expression was assessed in seven human bladder cancer cell lines. A replication-deficient recombinant adenoviral vector expressing shRNA targeting GPR87 (Ad-shGPR87), was constructed. Gene silencing was carried out using Ad-shGPR87. Immunohistochemical analysis was performed for transurethral resection of bladder tumor samples from 71 patients with non-muscle-invasive bladder cancer. We observed GPR87 expression in five of the seven cell lines, and silencing GPR87 gene expression significantly reduced cell viability. GPR87 expression was positive in 38 (54%) of 71 tumors. Ki-67 index was associated with positive GPR87 staining status (p < 0.0001). Patients with GPR87-positive tumors had shorter intravesical recurrence-free survival than those with GPR87-negative tumors (p = 0.010). Multivariate analysis revealed that GPR87 staining status was an independent prognostic parameter for intravesical recurrence (p = 0.041). Progression from non-muscle-invasive to muscle-invasive tumor was more frequently observed in patients with GPR87-positive tumors, although this trend did not reach statistical significance (p = 0.056). These results warrant further prospective studies to clarify the role of GPR87 expression in intravesical recurrence and progression in bladder cancer. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by loss of memory and cognitive capacity. Given the limitations to analyze brain cells, it is important to study whether peripheral lymphocytes can provide biological markers for AD, an interesting approach, once they represent the overall condition of the organism. To that extent, we sought to find whether lymphocytes of AD patients present DNA damage and repair kinetics different from those found in elderly matched controls (EC group) under in vitro treatment with hydrogen peroxide. We found that AD patient cells indeed showed an altered DNA repair kinetics (comet assay). Real-time quantitative analysis of genes associated with DNA stress response also showed that FANCG and CDKN1A are upregulated in AD, while MTH1 is downregulated, compared with the control group. In contrast, the expression of ATM, ATR and FEN1 genes does not seem to differ between these groups. Interestingly, TP53 protein expression was increased in AD patients. Therefore, we found that kinetics of the stress response in the DNA were significantly different in AD patients, supporting the hypothesis that repair pathways may be compromised in AD and that peripheral lymphocytes can reveal this condition. Full article

Ricin toxin binding subunit B (RTB) is one of the subunits of the ricin protein. RTB has been used as adjuvant, but little is known about its mechanism. In this study, we found that RTB increased not only nitric oxide (NO) release, but also tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in mouse macrophage cell line RAW264.7 cells. They subsequently exhibited enhanced ConA-induced T-cell and LPS-induced B-cell proliferative responses. We also examined the cytokines that were produced from splenocytes following in vitro RTB administration. Increased levels of IL-2, interferon (IFN)-γ and TNF-α were observed, while IL-4 and IL-5 were unaffected. These results demonstrate that recombinant RTB can act on the immune system and activate T-cells by introducing a Th1 immune response. Th1 cells might be the primary cellular target affected by RTB. Our results suggest that the recombinant RTB can promote the activation of macrophages and has a beneficial effect on immunomodulatory activity. Full article

The sequential sulfonylation/desulfination reactions of 5-benzylthiohydantoin with excess arylsulfonyl chlorides in the presence of triethylamine have been developed to afford a wide range of 5-arylidene thiohydantoin derivatives in moderate to excellent yields. A plausible sulfonylation/desulfination mechanism was proposed. The bioassay showed that these compounds exhibit certain fungicidal activities with the 71.9% inhibition rate of 2K against B. cinerea, and 57.6% inhibition rate of 2m against A. solani, respectively. Full article

Recurrent, metastatic prostate cancer continues to be a leading cause of cancer-death in men. The androgen receptor (AR) is a modular, ligand-inducible transcription factor that regulates the expression of genes that can drive the progression of this disease, and as a consequence, this receptor is a key therapeutic target for controlling prostate cancer. The current drugs designed to directly inhibit the AR are called anti-androgens, and all act by competing with androgens for binding to the androgen/ligand binding site. Unfortunately, with the inevitable progression of the cancer to castration resistance, many of these drugs become ineffective. However, there are numerous other regulatory sites on this protein that have not been exploited therapeutically. The regulation of AR activity involves a cascade of complex interactions with numerous chaperones, co-factors and co-regulatory proteins, leading ultimately to direct binding of AR dimers to specific DNA androgen response elements within the promoter and enhancers of androgen-regulated genes. As part of the family of nuclear receptors, the AR is organized into modular structural and functional domains with specialized roles in facilitating their inter-molecular interactions. These regions of the AR present attractive, yet largely unexploited, drug target sites for reducing or eliminating androgen signaling in prostate cancers. The design of small molecule inhibitors targeting these specific AR domains is only now being realized and is the culmination of decades of work, including crystallographic and biochemistry approaches to map the shape and accessibility of the AR surfaces and cavities. Here, we review the structure of the AR protein and describe recent advancements in inhibiting its activity with small molecules specifically designed to target areas distinct from the receptor’s androgen binding site. It is anticipated that these new classes of anti-AR drugs will provide an additional arsenal to treat castration-resistant prostate cancer. Full article

The TiO₂-montmorillonite (TiO₂-MMT) complex was prepared by blending TiO₂ sol and MMT with certain ratio, and its properties as an enzyme immobilization support were investigated. The pristine MMT and TiO₂-MMT calcined at 800 °C (TiO₂-MMT800) were used for comparison to better understand the immobilization mechanism. The structures of the pristine MMT, TiO₂-MMT, and TiO₂-MMT800 were examined by HR-TEM, XRD and BET. SEM was employed to study different morphologies before and after laccase immobilization. Activity and kinetic parameters of the immobilized laccase were also determined. It was found that the TiO₂ nanoparticles were successfully introduced into the MMT layer structure, and this intercalation enlarged the “d value” of two adjacent MMT layers and increased the surface area, while the calcination process led to a complete collapse of the MMT layers. SEM results showed that the clays were well coated with adsorbed enzymes. The study of laccase activity revealed that the optimum pH and temperature were pH = 3 and 60 °C, respectively. In addition, the storage stability for the immobilized laccase was satisfactory. The kinetic properties indicated that laccase immobilized on TiO₂-MMT complexes had a good affinity to the substrate. It has been proved that TiO₂-MMT complex is a good candidate for enzyme immobilization. Full article

Leafy gall is a plant hyperplasia induced upon Rhodococcus fascians infection. Previously, by genomic and transcriptomic analysis, it has been reported that, at the early stage of symptom development, both primary and secondary metabolisms are modified. The present study is based on the hypothesis that fully developed leafy gall, could represent a potential source of new bioactive compounds. Therefore, non-targeted metabolomic analysis of aqueous and chloroform extracts of leafy gall and non-infected tobacco was carried out by ¹H-NMR coupled to principal component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). Polar metabolite profiling reflects modifications mainly in the primary metabolites and in some polyphenolics. In contrast, main modifications occurring in non-polar metabolites concern secondary metabolites, and gas chromatography and mass spectrometry (GC-MS) evidenced alterations in diterpenoids family. Analysis of crude extracts of leafy galls and non-infected tobacco leaves exhibited a distinct antiproliferative activity against all four tested human cancer cell lines. A bio-guided fractionation of chloroformic crude extract yield to semi-purified fractions, which inhibited proliferation of glioblastoma U373 cells with IC₅₀ between 14.0 and 2.4 µg/mL. Discussion is focused on the consequence of these metabolic changes, with respect to plant defense mechanisms following infection. Considering the promising role of diterpenoid family as bioactive compounds, leafy gall may rather be a propitious source for drug discovery. Full article

Two clinical forms of functional dyspepsia (FD) are listed in the Rome III criteria: postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS), differing in the recurrence of ailments depending on the diet. Continuous EPS (CEPS) is observed in some EPS patients, also at night, but its cause is still unknown. We showed previously that melatonin (MEL) homeostasis may be associated with FD. In the present work we evaluated selected components of melatonin homeostasis in patients with CEPS. The study included 30 patients with CEPS, 21 women and nine men, aged 21–49 years and 30 control subjects (EPS excluded); organic and mental diseases, as well as Helicobacter pylori infection, were excluded in both groups. The average severity of abdominal pain in the last three months was estimated in a 10-point scale (Visual Analog Scale). The levels of mRNA expression of arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), the main components of MEL homeostasis, were determined in gastric mucosa with real time PCR. The fasting serum level of MEL (at 09:00 a.m.) and circadian urine excretion of 6-sulfatoxymelatonin (6-HMS) were determined with ELISA. AANAT expression in antral mucosa of control subjects was 1.76 ± 0.41, in the gastric body 1.35 ± 0.38, and in the dyspeptic group 1.42 ± 0.38 (p < 0.05) and 0.92 ± 0.55 (p < 0.05), respectively. HIOMT expression in the control was 2.05 ± 0.70 in the antrum and 1.57 ± 0.69 in the body and in the CEPS group, it was: 1.51 ± 0.57 (p < 0.05) and 0.74 ± 0.31 (p < 0.001), respectively. MEL concentration (pg/mL) was 9.41 ± 3.09 in the control group and 5.62 ± 1.34 (p < 0.01) in the CEPS group. Urinary 6-HMS excretion (μg/24 h) was 11.40 ± 4.46 in the controls and 7.68 ± 2.88 (p < 0.05) in the CEPS. Moreover, a negative correlation was found between the tested parameters and severity of epigastric pain. These results indicate that patients with CEPS may display low level of AANAT and HIOMT expression in gastric mucosa, resulting in decreased MEL synthesis. Full article

Total parenteral nutrition (TPN) is an artificial way to support daily nutritional requirements by bypassing the digestive system, but long-term TPN administration may cause severe liver dysfunction. Glycyrrhizin is an active component of licorice root that has been widely used to treat chronic hepatitis. The aim of this study is to investigate the hepatoprotective effect of glycyrrhizin on TPN-associated acute liver injury in vivo. Liver dysfunction was induced by intravenous infusion of TPN at a flow rate of 20 mL/kg/h for three h in Sprague Dawley rats. The rats were pretreated with Glycyrrhizin (1, 3 and 10 mg/kg intravenously). After receiving TPN or saline (control group) for three h, the rats were sacrificed, blood samples were collected for biochemical analyses and liver tissue was removed for histopathological and immunohistochemical examination. We found that aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB) and triglyceride (TG) levels were significantly increased in the TPN group without glycyrrhizin pretreatment and decreased in the glycyrrhizin-pretreated TPN group in a dose-dependent manner. The stained liver sections showed that glycyrrhizin relieved acute liver injury. The upregulation of serum protein biomarkers of reactive nitrogen species, including nitrotyrosine and inducible NO synthase (iNOS), were attenuated by glycyrrhizin pretreatment. Levels of endoplasmic reticulum (ER) stress factors, such as phosphorylation of JNK1/2, p38 MAPK and CHOP, were decreased by glycyrrhizin pretreatment. In summary, our results suggest that glycyrrhizin decreases TPN-associated acute liver injury factors by suppressing endoplasmic reticulum stress and reactive nitrogen stress. Full article

Two new norsesquiterpenoids, solanerianones A and B (1–2), together with nine known compounds, including four sesquiterpenoids, (−)-solavetivone (3), (+)-anhydro-β-rotunol (4), solafuranone (5), lycifuranone A (6); one alkaloid, N-trans-feruloyltyramine (7); one fatty acid, palmitic acid (8); one phenylalkanoid, acetovanillone (9), and two steroids, β-sitosterol (10) and stigmasterol (11) were isolated from the n-hexane-soluble part of the roots of Solanum erianthum. Their structures were elucidated on the basis of physical and spectroscopic data analyses. The anti-inflammatory activity of these isolates was monitored by nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells. The cytotoxicity towards human lung squamous carcinoma (CH27), human hepatocellular carcinoma (Hep 3B), human oral squamous carcinoma (HSC-3) and human melanoma (M21) cell lines was also screened by using an MTT assay. Of the compounds tested, 3 exhibited the strongest NO inhibition with the average maximum inhibition (E_max) at 100 μM and median inhibitory concentration (IC₅₀) values of 98.23% ± 0.08% and 65.54 ± 0.18 μM, respectively. None of compounds (1–9) was found to possess cytotoxic activity against human cancer cell lines at concentrations up to 30 μM. Full article

Two studies were conducted to determine the effects of dietary cholesterol (CHO) and cholesterol oxides (COPs) on the development of atherosclerosis and the changes in fatty acid and blood characteristics in rabbits. In the first study, forty male New Zealand white rabbits were divided into 5 groups and fed commercial rabbit chow with no added CHO or COPs, 1 g CHO, 0.9 g CHO + 0.1 g COPs, 0.8 g CHO + 0.2 g COPs, or 0.5 g CHO + 0.5 g COPs per kg diet. In the second study, 24 male New Zealand White rabbits were divided into 3 groups and fed a diet containing 2 g CHO, 1.6 g CHO + 0.4 g COPs, or 1.2 g CHO + 0.8 g COPs per kg diet. All diets induced atherosclerotic lesions in the rabbits’ ascending thoracic aorta. The serum CHO and triglyceride levels (p < 0.05) increased significantly with the increased levels of CHO in the diets. Dietary CHO or COPs did not influence high-density lipoprotein CHO levels. The ratio of saturated fatty acid to unsaturated fatty acid increased as the level of dietary CHO and COPs increased. Full article

Human chorionic gonadotropin beta subunit (CGB) is a marker of pregnancy as well as trophoblastic and nontrophoblastic tumors. CGB is encoded by a cluster of six genes, of which type II genes (CGB3/9, 5 and 8) have been shown to be upregulated in relation to type I genes (CGB6/7) in both placentas and tumors. Recent studies revealed that CGB1 and CGB2, originally considered as pseudogenes, might also be active, however, the protein products of these genes have not yet been identified. Our study demonstrates the presence of CGB1 and CGB2 transcripts in ovarian carcinomas. While CGB1 and CGB2 gene activation was not detected in normal ovaries lacking cancerous development, our study demonstrates the presence of CGB1 and CGB2 transcripts in 41% of analyzed ovarian cancer cases. Full article

Over expressing in PTPN1 (encoding Protein tyrosine phosphatase 1B, PTP1B), a protein tyrosine phosphatase (PTP) that plays an overall positive role in insulin signaling, is linked to the pathogenesis of diabetes and obesity. The relationship between PTP1B and human diseases exhibits PTP1B as the target to treat these diseases. In this article, small weight molecules of the imidazolidine series were screened from databases and optimized on silicon as the inhibitors of PTP1B based on the steric conformation and electronic configuration of thiazolidinedione (TZD) compounds. The top three candidates were tested using an in vitro biological assay after synthesis. Finally, we report a novel inhibitor, Compound 13, that specifically inhibits PTP1B over the closely related phosphatase Src homology 2 (SH2) domain-containing phosphatase 2 (SHP-2) at 80 μΜ. Its IC₅₀ values are reported in this paper as well. This compound was further verified by computer analysis for its ability to combine the catalytic domains of PTP1B and SHP-2 by molecular dynamics (MD) simulations. Full article

Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD), that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD), which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of the SBD of the bacterial Hsp70, DnaK, consists of two sub-domains: A β-sandwich part containing the hydrophobic cavity to which the hepta-peptide NRLLLTG (NR) is bound, and a segment made of 5 α-helices, called the “lid” that caps the top of the β-sandwich domain. In the present study we used the Escherichia coli Hsp70, DnaK, as a model for Hsp70 proteins, focusing on its SBD domain, examining the changes in the lid conformation. We deliberately decoupled the NBD from the SBD, limiting the study to the structure of the SBD section, with an emphasis on the interaction between the charges of the peptide with the residues located in the lid. Molecular dynamics simulations of the complex revealed significant mobility within the lid structure; as the structure was released from the forces operating during the crystallization process, the two terminal helices established a contact with the positive charge at the tip of the peptide. This contact is manifested only in the presence of electrostatic attraction. The observed internal motions within the lid provide a molecular role for the function of this sub-domain during the reaction cycle of Hsp 70 chaperones. Full article

The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) from nano-composite scaffolds (PLGA/PCL/nHA) loaded with vascular stents (PLCL/Col/nHA) for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs), bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA). AlamarBlue assay and alkaline phosphatase (ALP) activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01). In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large bone defects in bone tissue engineering. Full article

Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc. Full article

AhAREB1 (Arachis hypogaea Abscisic-acid Response Element Binding Protein 1) is a member of the basic domain leucine zipper (bZIP)-type transcription factor in peanut. Previously, we found that expression of AhAREB1 was specifically induced by abscisic acid (ABA), dehydration and drought. To understand the drought defense mechanism regulated by AhAREB1, transgenic Arabidopsis overexpressing AhAREB1 was conducted in wild-type (WT), and a complementation experiment was employed to ABA non-sensitivity mutant abi5 (abscisic acid-insensitive 5). Constitutive expression of AhAREB1 confers water stress tolerance and is highly sensitive to exogenous ABA. Microarray and further real-time PCR analysis revealed that drought stress, reactive oxygen species (ROS) scavenging, ABA synthesis/metabolism-related genes and others were regulated in transgenic Arabidopsis overexpressing AhAREB1. Accordingly, low level of ROS, but higher ABA content was detected in the transgenic Arabidopsis plants’ overexpression of AhAREB1. Taken together, it was concluded that AhAREB1 modulates ROS accumulation and endogenous ABA level to improve drought tolerance in transgenic Arabidopsis. Full article

Primitive proteins are proposed to have utilized organic cofactors more frequently than transition metals in redox reactions. Thus, an experimental validation on whether a protein constituted solely by early amino acids and an organic cofactor can perform electron transfer activity is an urgent challenge. In this paper, by substituting “late amino acids (C, F, M, T, W, and Y)” with “early amino acids (A, L, and V)” in a flavodoxin, we constructed a flavodoxin mutant and evaluated its characteristic properties. The major results showed that: (1) The flavodoxin mutant has structural characteristics similar to wild-type protein; (2) Although the semiquinone and hydroquinone flavodoxin mutants possess lower stability than the corresponding form of wild-type flavodoxin, the redox potential of double electron reduction E_m,7 (fld) reached −360 mV, indicating that the flavodoxin mutant constituted solely by early amino acids can exert effective electron transfer activity. Full article

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories. Full article

An attempt was made to use functionalized graphene oxide (GO) to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene), a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC)-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable. Full article

In this work, we assessed the effects of sinomenine (SN) on intestinal octreotide (OCT) absorption both in Caco-2 cell monolayers and in rats. We also investigated the molecular mechanisms of tight junction (TJ) disruption and recovery by SN-mediated changes in the claudin-1 and protein kinase C (PKC) signaling pathway. The data showed that exposure to SN resulted in a significant decrease in the expression of claudin-1, which represented TJ weakening and paracellular permeability enhancement. Then, the recovery of TJ after SN removal required an increase in claudin-1, which demonstrated the transient and reversible opening for TJ. Meanwhile, the SN-mediated translocation of PKC-α from the cytosol to the membrane was found to prove PKC activation. Finally, SN significantly improved the absolute OCT bioavailability in rats and the transport rate in Caco-2 cell monolayers. We conclude that SN has the ability to enhance intestinal OCT absorption and that these mechanisms are related at least in part to the important role of claudin-1 in SN-mediated, reversible TJ opening via PKC activation. Full article

Adjusting the inter-particle distances in ordered nanoparticle arrays can create new nano-devices and is of increasing importance to a number of applications such as nanoelectronics and optical devices. The assembly of negatively charged polystyrene (PS) nanoparticles (NPs) on Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes, quaternized PDMAEMA brushes and Si/PEI/(PSS/PAH)2, was studied using dip- and spin-coating techniques. By dip-coating, two dimensional (2-D), randomly distributed non-close packed particle arrays were assembled on Si/PEI/(PSS/PAH)2 and PDMAEMA brushes. The inter-particle repulsion leads to lateral mobility of the particles on these surfaces. The 200 nm diameter PS NPs tended to an inter-particle distance of 350 to 400 nm (center to center). On quaternized PDMAEMA brushes, the strong attractive interaction between the NPs and the brush dominated, leading to clustering of the particles on the brush surface. Particle deposition using spin-coating at low spin rates resulted in hexagonal close-packed multilayer structures on Si/PEI/(PSS/PAH)2. Close-packed assemblies with more pronounced defects are also observed on PDMAEMA brushes and QPDMAEMA brushes. In contrast, randomly distributed monolayer NP arrays were achieved at higher spin rates on all polyelectrolyte architectures. The area fraction of the particles decreased with increasing spin rate. Full article

Poly-(R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues), which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100–200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA) of Escherichia coli. Full article

The Src gene product (Src) and the epidermal growth factor receptor (EGFR) are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845) in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases) or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase). A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation. Full article

Penile carcinoma (PeCa) represents an important public health problem in poor and developing countries. Despite its unpredictable behavior and aggressive treatment, there have only been a few reports regarding its molecular data, especially epigenetic mechanisms. The functional diversity in different cell types is acquired by chromatin modifications, which are established by epigenetic regulatory mechanisms involving DNA methylation, histone acetylation, and miRNAs. Recent evidence indicates that the dysregulation in these processes can result in the development of several diseases, including cancer. Epigenetic alterations, such as the methylation of CpGs islands, may reveal candidates for the development of specific markers for cancer detection, diagnosis and prognosis. There are a few reports on the epigenetic alterations in PeCa, and most of these studies have only focused on alterations in specific genes in a limited number of cases. This review aims to provide an overview of the current knowledge of the epigenetic alterations in PeCa and the promising results in this field. The identification of epigenetically altered genes in PeCa is an important step in understanding the mechanisms involved in this unexplored disease. Full article

By the age of 80, approximately 80% of men will manifest some cancerous cells within their prostate, indicating that prostate cancer constitutes a major health burden. While this disease is clinically insignificant in most men, it can become lethal in others. The most challenging task for clinicians is developing a patient-tailored treatment in the knowledge that this disease is highly heterogeneous and that relatively little adequate prognostic tools are available to distinguish aggressive from indolent disease. Next-generation sequencing allows a description of the cancer at an unprecedented level of detail and at different levels, going from whole genome or exome sequencing to transcriptome analysis and methylation-specific immunoprecipitation, followed by sequencing. Integration of all these data is leading to a better understanding of the initiation, progression and metastatic processes of prostate cancer. Ultimately, these insights will result in a better and more personalized treatment of patients suffering from prostate cancer. The present review summarizes current knowledge on copy number changes, gene fusions, single nucleotide mutations and polymorphisms, methylation, microRNAs and long non-coding RNAs obtained from high-throughput studies. Full article

Epidermis is one of the best-studied tissues in mammals that contain types of stem cells. Outstanding works in recent years have shed great light on behaviors of different epidermal stem cell populations in the homeostasis and regeneration of the epidermis as well as hair follicles. Also, the molecular mechanisms governing these stem cells are being elucidated, from genetic to epigenetic levels. Compared with the explicit knowledge about adult skin, embryonic development of the epidermis, especially the early period, still needs exploration. Furthermore, stem cells in the embryonic epidermis are largely unstudied or ambiguously depicted. In this review, we will summarize and discuss the process of embryonic epidermal development, with focuses on some key molecular regulators and the role of the sub-epidermal mesenchyme. We will also try to trace adult epidermal stem cell populations back to embryonic development. In addition, we will comment on in vitro derivation of epidermal lineages from ES cells and iPS cells. Full article

Lignin and cellulose represent the two main components of plant secondary walls and the most abundant polymers on Earth. Quantitatively one of the principal products of the phenylpropanoid pathway, lignin confers high mechanical strength and hydrophobicity to plant walls, thus enabling erect growth and high-pressure water transport in the vessels. Lignin is characterized by a high natural heterogeneity in its composition and abundance in plant secondary cell walls, even in the different tissues of the same plant. A typical example is the stem of fibre crops, which shows a lignified core enveloped by a cellulosic, lignin-poor cortex. Despite the great value of fibre crops for humanity, however, still little is known on the mechanisms controlling their cell wall biogenesis, and particularly, what regulates their spatially-defined lignification pattern. Given the chemical complexity and the heterogeneous composition of fibre crops’ secondary walls, only the use of multidisciplinary approaches can convey an integrated picture and provide exhaustive information covering different levels of biological complexity. The present review highlights the importance of combining high throughput -omics approaches to get a complete understanding of the factors regulating the lignification heterogeneity typical of fibre crops. Full article

Pineal gland melatonin is the darkness hormone, while extra-pineal melatonin produced by the gonads, gut, retina, and immune competent cells acts as a paracrine or autocrine mediator. The well-known immunomodulatory effect of melatonin is observed either as an endocrine, a paracrine or an autocrine response. In mammals, nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) blocks noradrenaline-induced melatonin synthesis in pinealocytes, which induces melatonin synthesis in macrophages. In addition, melatonin reduces NF-κB activation in pinealocytes and immune competent cells. Therefore, pathogen- or danger-associated molecular patterns transiently switch the synthesis of melatonin from pinealocytes to immune competent cells, and as the response progresses melatonin inhibition of NF-κB activity leads these cells to a more quiescent state. The opposite effect of NF-κB in pinealocytes and immune competent cells is due to different NF-κB dimers recruited in each phase of the defense response. This coordinated shift of the source of melatonin driven by NF-κB is called the immune-pineal axis. Finally, we discuss how this concept might be relevant to a better understanding of pathological conditions with impaired melatonin rhythms and hope it opens new horizons for the research of side effects of melatonin-based therapies. Full article

Prostate cancer (PCa) is a leading cause of cancer-related death of men globally. Since its introduction, there has been intense debate as to the effectiveness of the prostate specific antigen (PSA) test as a screening tool for PCa. It is now evident that the PSA test produces unacceptably high rates of false positive results and is not prognostic. Here we review the current status of molecular biomarkers that promise to be prognostic and that might inform individual patient management. It highlights current efforts to identify biomarkers obtained by minimally invasive methods and discusses current knowledge with regard to gene fusions, mRNA and microRNAs, immunology, and cancer-associated microparticles. Full article

Melatonin receptors are members of the G protein-coupled receptor (GPCR) family. Three genes for melatonin receptors have been cloned. The MT1 (or Mel1a or MTNR1A) and MT2 (or Mel1b or MTNR1B) receptor subtypes are present in humans and other mammals, while an additional melatonin receptor subtype, Mel1c (or MTNR1C), has been identified in fish, amphibians and birds. Another melatonin related orphan receptor, GPR50, which does not bind melatonin, is found exclusively in mammals. The hormone melatonin is secreted primarily by the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone acts systemically in numerous organs. In the brain, it is involved in the regulation of various neural and endocrine processes, and it readjusts the circadian pacemaker, the suprachiasmatic nucleus. This article reviews recent studies of gene organization, expression, evolution and mutations of melatonin receptor genes of vertebrates. Gene polymorphisms reveal that numerous mutations are associated with diseases and disorders. The phylogenetic analysis of receptor genes indicates that GPR50 is an outgroup to all other melatonin receptor sequences. GPR50 may have separated from a melatonin receptor ancestor before the split between MTNR1C and the MTNR1A/B ancestor. Full article

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions. Full article